P341: L-ASPARAGINASE BY-PRODUCT GLUTAMATE IMPEDES THE EFFECT OF CHEMOTHERAPY BY AUGMENTING GLUTATHIONE BIOSYNTHESIS
Authors: Katerina Hlozkova, Maryna Vasylkivska, Martina Zwyrtkova, Matus Kolarik, Eliska Potuckova, Natividad Alquezar Artieda, Marketa Zaliova, Jan Trka, Daniel Tennant, Julia Starkova
Journal: HemaSphere (2023)

Background: L-asparaginase (ASNase) is one of the crucial components of acute lymphoblastic leukemia (ALL) therapy. Therapeutic ASNase concentration transforms asparagine (Asn) and glutamine (Gln) to aspartate (Asp) and glutamate (Glu), respectively. High Asp and Glu extracellular concentrations are considered to be a by-product of ASNase treatment without any known consequences. However, our results show that even though TCA cycle, the important producer of Asp and Glu, is diminished after ASNase treatment, leukemic cells are able to maintain intracellular Asp and Glu levels. Aims: The main aim is to elucidate the mechanism by which leukemic cells are able to maintain Asp and Glu levels. The second aim is to evaluate the role of Asp and Glu in the resistance mechanism of leukemic cells to ASNase. Methods: Stable isotope tracing was used to measure Asp and Glu import and activity of glutathione (GSH) synthesis. Growth and viability of leukemic cells under different nutrient conditions were determined by flow cytometry. Reactive oxygen species (ROS) production was measured using H2DCFDA on flow cytometry. Results: Using U13C-Asp and U13C-Glu we discovered that NALM6 and REH cell lines were able to import Asp and Glu from the culture media. Noteworthy, leukemia cell lines and also primary leukemia cells expressed the genes coding for Asp/Glu transporters. Moreover, inhibitor of Asp/Glu transporters moderately sensitized NALM6 cell line to ASNase. Next, we broadened the number of ALL cell lines and discovered that high Glu doses, but not high Asp doses, helped the cells to survive in Asn-/Gln-depleted conditions. Furthermore, in high Glu, but not in high Asp conditions, we detected elevated intracellular ROS compared to Asn-/Gln-depleted media. Since GSH is the main antioxidant molecule in the cells and Glu is one of its three amino acids, we pursued the idea that cells under Asn-/Gln-depleted conditions use Glu to synthesize GSH and by that overcome the nutrient stress. Indeed, we discovered that both NALM6 and REH cells used imported Glu to biosynthesize GSH. Preliminary data with ROS scavenger showed that ROS scavenging further improved leukemia cell proliferation in high Glu conditions. Together with the fact that imported Glu also fueled TCA cycle, GSH biosynthesis probably partially rescues the cells from oxidative stress caused by respiration. When we used GSH synthesis inhibitor BSO, proliferation of NALM6 cells was significantly more affected in high Glu condition compared to media without Glu. We did not see the same trend in REH cells, however, our results indicate that REH cells are more dependent on Glu anaplerosis compared to NALM6 cells in high Glu condition. Summary/Conclusion: This is the first study describing the transport of Glu into leukemic cells and its survival advantage after ASNase treatment. This work was supported by AZV grant NU20J-03-00032 and NU22-07-00087. Keywords: Resistance, Acute lymphoblastic leukemia, L-asparaginase, Antioxidants